ABSTRACT The ribosome can functionally respond to specific nascent peptides. Such response, which can be additionally affected by small molecular weight cofactors, is a key component of the mechanism of control of important bacterial and eukaryotic genes. However, the molecular mechanisms of recognition of the regulatory nascent peptide and cofactor and the basic principles of the ribosomal response are poorly understood. Expression of a number of inducible antibiotic resistance genes in bacterial pathogens is controlled by nascent peptide-dependent programmed translation arrest promoted by antibiotic. Recently, a distinct class of regulatory nascent peptides encoded in the regulatory cistrons of many macrolide resistance genes has been identified. The peptides of this class are characterize dby the presence of an 'RLR' (Arg-Leu-Arg) motif. Preliminary studies showed that the mechanism of programmed translation arrest directed by these peptides is principally different from all of the previously studied systems. Furthermore, nascent peptides as short as three or four amino acids were found to be sufficient to direct drug-dependent ribosome arrest thereby yielding the shortest known stalling peptides. Such minimalistic stalling nascent peptides and the knowledge of the exact binding site of the stalling cofactor (the inducing antibiotic) provide unprecedented tools for unraveling important fundamental aspects of the general mechanisms of nascent peptide- and small cofactor-controlled programmed translation arrest. In the three specific aims of this proposal we will 1) explore the structural features of the minimalist nascent peptide required for directing ribosome stalling, 2) analyze the role of the antibiotic cofactor in programmed translation arrest, and 3) unravel the mechanism of the stalling signal recognition and structural features of the ribosome in the arrested state. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE One of the major challenges of modern medicine is combating the spread of resistance to antibiotics. Many bacterial pathogens activate expression of the resistance genes in response to the presence of antibiotics. Understanding the molecular mechanisms of inducible resistance will, among other applications, pave the way for novel strategies to develop superior drugs capable of curbing bacterial infections.